poolMC: Smart pooling of mRNA samples in microarray experiments

Background: Typically, pooling of mRNA samples in microarray experiments implies mixing mRNA from several biological-replicate samples before hybridization onto a microarray chip. Here we describe an alternative smart pooling strategy in which different samples, not necessarily biological replicates, are pooled in an information theoretic efficient way. Further, each sample is tested on multiple chips, but always in pools made up of different samples. The end goal is to exploit the compressibility of microarray data to reduce the number of chips used and increase the robustness to noise in measurements. Results: A theoretical framework to perform smart pooling of mRNA samples in microarray experiments was established and the software implementation of the pooling and decoding algorithms was developed in MATLAB. A proof-of-concept smart pooled experiment was performed using validated biological samples on commercially available gene chips. Conclusions: The theoretical developments and experimental demonstration in this paper provide a useful starting point to investigate smart pooling of mRNA samples in microarray experiments. Important conditions for its successful implementation include linearity of measurements, sparsity in data, and large experiment size.

A mechanistic framework for auxin dependent Arabidopsis root hair elongation to low external phosphate

Phosphate (P) is an essential macronutrient for plant growth. Roots employ adaptive mechanisms to forage for P in soil. Root hair elongation is particularly important since P is immobile. Here we report that auxin plays a critical role promoting root hair growth in Arabidopsis in response to low external P. Mutants disrupting auxin synthesis (taa1) and transport (aux1) attenuate the low P root hair response. Conversely, targeting AUX1 expression in lateral root cap and epidermal cells rescues this low P response in aux1. Hence auxin transport from the root apex to differentiation zone promotes auxin-dependent hair response to low P. Low external P results in induction of root hair expressed auxin-inducible transcription factors ARF19, RSL2, and RSL4. Mutants lacking these genes disrupt the low P root hair response. We conclude auxin synthesis, transport and response pathway components play critical roles regulating this low P root adaptive response

Conserved Roles of CrRLK1L Receptor-Like Kinases in Cell Expansion and Reproduction from Algae to Angiosperms

Receptor-like kinases (RLKs) are regulators of plant development through allowing cells to sense their extracellular environment. They facilitate detection of local endogenous signals, in addition to external biotic and abiotic stimuli. The Catharanthus roseus RLK1-like (CrRLK1L) protein kinase subfamily, which contains FERONIA, plays a central role in regulating fertilization and in cell expansion mechanisms such as cell elongation and tip growth, as well as having indirect links to plant–pathogen interactions. Several components of CrRLK1L signaling pathways have been identified, including an extracellular ligand, coreceptors, and downstream signaling elements. The presence and abundance of the CrRLK1L proteins in the plant kingdom suggest an origin within the Streptophyta lineage, with a notable increase in prevalence in the seeded land plants. Given the function of the sole CrRLK1L protein in a charophycean alga, the possibility of a conserved role in detection and/or regulation of cell wall integrity throughout the Strephtophytes is discussed. Orthologs of signaling pathway components are also present in extant representatives of non-vascular land plants and early vascular land plants including the liverwort Marchantia polymorpha, the moss Physcomitrella patens and the lycophyte Selaginella moellendorffii. Deciphering the roles in development of the CrRLK1L protein kinases in early diverging land plants will provide insights into their ancestral function, furthering our understanding of this diversified subfamily of receptors in higher plants

Multi-omics analysis identifies genes mediating the extension of cell walls in the Arabidopsis thaliana root elongation zone

Plant cell wall composition is important for regulating growth rates, especially in roots. However, neither analyses of cell wall composition nor transcriptomes on their own can comprehensively reveal which genes and processes are mediating growth and cell elongation rates. This study reveals the benefits of carrying out multiple analyses in combination. Sections of roots from five anatomically and functionally defined zones in Arabidopsis thaliana were prepared and divided into three biological replicates. We used glycan microarrays and antibodies to identify the major classes of glycans and glycoproteins present in the cell walls of these sections, and identified the expected decrease in pectin and increase in xylan from the meristematic zone (MS), through the rapid and late elongation zones (REZ, LEZ) to the maturation zone and the rest of the root, including the emerging lateral roots. Other compositional changes included extensin and xyloglucan levels peaking in the REZ and increasing levels of arabinogalactan-proteins (AGP) epitopes from the MS to the LEZ, which remained high through the subsequent mature zones. Immuno-staining using the same antibodies identified the tissue and (sub)cellular localization of many epitopes. Extensins were localized in epidermal and cortex cell walls, while AGP glycans were specific to different tissues from root-hair cells to the stele. The transcriptome analysis found several gene families peaking in the REZ. These included a large family of peroxidases (which produce the reactive oxygen species (ROS) needed for cell expansion), and three xyloglucan endo-transglycosylase/hydrolase genes (XTH17, XTH18, and XTH19). The significance of the latter may be related to a role in breaking and re-joining xyloglucan cross-bridges between cellulose microfibrils, a process which is required for wall expansion. Knockdowns of these XTHs resulted in shorter root lengths, confirming a role of the corresponding proteins in root extension growth

A Gene Regulatory Network for Root Epidermis Cell Differentiation in Arabidopsis

The root epidermis of Arabidopsis provides an exceptional model for studying the molecular basis of cell fate and differentiation. To obtain a systems-level view of root epidermal cell differentiation, we used a genome-wide transcriptome approach to define and organize a large set of genes into a transcriptional regulatory network. Using cell fate mutants that produce only one of the two epidermal cell types, together with fluorescence-activated cell-sorting to preferentially analyze the root epidermis transcriptome, we identified 1,582 genes differentially expressed in the root-hair or non-hair cell types, including a set of 208 “core” root epidermal genes. The organization of the core genes into a network was accomplished by using 17 distinct root epidermis mutants and 2 hormone treatments to perturb the system and assess the effects on each gene's transcript accumulation. In addition, temporal gene expression information from a developmental time series dataset and predicted gene associations derived from a Bayesian modeling approach were used to aid the positioning of genes within the network. Further, a detailed functional analysis of likely bHLH regulatory genes within the network, including MYC1, bHLH54, bHLH66, and bHLH82, showed that three distinct subfamilies of bHLH proteins participate in root epidermis development in a stage-specific manner. The integration of genetic, genomic, and computational analyses provides a new view of the composition, architecture, and logic of the root epidermal transcriptional network, and it demonstrates the utility of a comprehensive systems approach for dissecting a complex regulatory network

Microstructured Hydrogel Templates for the Formation of Conductive Gold Nanowire Arrays

Microstructured hydrogel allows for a new template-guided method to obtain conductive nanowire arrays on a large scale. To generate the template, an imprinting process is used in order to synthesize the hydrogel directly into the grooves of wrinkled polydimethylsiloxane (PDMS). The resulting poly(N-vinylimidazole)-based hydrogel is defined by the PDMS stamp in pattern and size. Subsequently, tetrachloroaurate(III) ions from aqueous solution are coordinated within the humps of the N-vinylimidazole-containing polymer template and reduced by air plasma. After reduction and development of the gold, to achieve conductive wires, the extension perpendicular to the long axis (width) of the gold strings is considerably reduced compared to the dimension of the parental hydrogel wrinkles (from approximate to 1 mu m down to 200-300 nm). At the same time, the wire-to-wire distance and the overall length of the wires is preserved. The PDMS templates and hydrogel structures are analyzed with scanning force microscopy (SFM) and the gold structures via scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy. The conductivity measurements of the gold nanowires are performed in situ in the SEM, showing highly conductive gold leads. Hence, this method can be regarded as a facile nonlithographic top-down approach from micrometer-sized structures to nanometer-sized features

Fine-tuning of root elongation by ethylene: a tool to study dynamic structure-function relationships between root architecture and nitrate absorption

Background Recently developed genetic and pharmacological approaches have been used to explore NO3- /ethylene signalling interactions and how the modifications in root architecture by pharmacological modulation of ethylene biosynthesis affect nitrate uptake. Key Results Structure-function studies combined with recent approaches to chemical genomics highlight the non-specificity of commonly used inhibitors of ethylene biosynthesis such as AVG (L-aminoethoxyvinylglycine). Indeed, AVG inhibits aminotransferases such as ACC synthase (ACS) and tryptophan aminotransferase (TAA) involved in ethylene and auxin biosynthesis but also some aminotransferases implied in nitrogen (N) metabolism. In this framework, it can be assumed that the products of nitrate assimilation and hormones may interact through a hub in carbon (C) and N metabolism to drive the root morphogenetic programme (RMP). Although ethylene/auxin interactions play a major role in cell division and elongation in root meristems, shaping of the root system depends also on energetic considerations. Based on this finding, the analysis is extended to nutrient ion-hormone interactions assuming a fractal or constructal model for root development. Conclusion Therefore, the tight control of root structure-function in the RMP may explain why over-expressing nitrate transporter genes to decouple structure-function relationships and improve nitrogen use efficiency (NUE) has been unsuccessful